Mass spectrometry (MS) is a well known technique for obtaining qualitative and quantitative information from a sample. It is commonly used to determine molecular weight, identify chemical structures and accurately determine the composition of mixtures. Mass spectrometry is becoming increasingly important in biological research to determine the structure of organic molecules based upon the fragmentation pattern of ions formed when sample molecules are ionized. Using mass spectrometry, individual molecules of a sample are weighed by ionizing the molecules and measuring the trajectory of their response in a vacuum to various electric and magnetic fields. However, traditional techniques, such as electron ionization or the evaporation process associated with classical chemical ionization, often damage the molecule under analysis, thereby severely limiting the compounds which can be analyzed by mass spectrometry.
Improved ionization techniques have been developed, such as fast atom bombardment, thermospray, electrospray, and atmospheric pressure chemical ionization which produce intact molecular ions from high molecular weight, ionic and thermally labile molecules. As a result, mass spectrometry has become increasingly important for many new applications such as biological research, where detection and characterization of high molecular weight molecules is required.
Many different kinds of mass spectrometry are known in the art. Quadrupole mass spectrometry is commonly used in conjunction with electrospray. Although quadrupole systems provide good sensitivity, they are useful only for a limited mass range. Similarly, magnetic sector mass spectrometry systems are frequently used; although they provide accurate mass information, they have poor sensitivity.
Time-of-flight (TOF) mass spectrometers separate ions according to their mass-to-charge ratio by measuring the time it takes generated ions to travel to a detector. Time-of-flight mass spectrometers are advantageous because they are relatively simple, inexpensive instruments with virtually unlimited mass-to-charge ratio range. They have potentially higher sensitivity than scanning instruments because they can record all the ions generated from each ionization event. Time-of-flight mass spectrometers are particularly useful for measuring the mass-to-charge ratio of large organic molecules where conventional magnetic field mass spectrometers lack sensitivity. See, for example, U.S. Pat. Nos. 5,045,694, 5,160,840 and U.S. Ser. Nos. 08/488,127 and 08/446,544, specifically incorporated by reference.
Mass spectrometers include an ionization source for generating ions from the sample material under investigation. The ionization source contains one or more electrodes or electrostatic lenses for accelerating and properly directing an ion beam. Electrospray ionization is frequently used to obtain molecular weight information on large biopolymers, such as proteins. In electrospray, a sample solution containing molecules of interest is directed through a capillary tube and into an electrospray chamber. The end of the capillary tube is connected to a high voltage source and a voltage is applied to generate a fine spray of charged droplets. The droplets may be sprayed into a chamber and then introduced into a mass spectrometer for analysis.
These prior electrospray techniques, however, are only able to accommodate a very narrow range of liquid flow rates, generally in the range of microliters per minute. Thus, to accommodate faster flow rates it is necessary to render the flow rates compatible with droplet formation, ion creation and isolation processes.
Additionally, with larger liquid flow rates, such as those often associated with liquid chromatography, it is very difficult, if not impossible, to generate a spray of droplets by electrospray alone without a pneumatic assist, use of splitter and/or the addition of heat.
Systems for combining mass spectrometry and liquid chromatography have been described. (See, for example, U.S. Pat. No. 4,209,696). In these systems, carrier liquid from a liquid chromatograph is electrosprayed or pneumatically assisted electrosprayed and then analyzed by mass spectrometry. Unfortunately, these systems suffer significant limitations due to incompatible flow rates, and the complexity of splitters necessary for analysis or separation. Moreover, substantially all liquid chromatography effluents contain some non-volatile material, typically in the form of buffers, impurities, or sample residue. When a liquid chromatography solvent is vaporized, this non-volatile material is deposited on the interior of the mass spectrometer, causing a reduction in performance. Accordingly, a major problem with liquid chromatography/mass spectrometer interfaces is the disposal of solvent vapor, which, in addition to instrument contamination, produces hazardous organic vapor. Devices of the art typically attempt to address this problem by supplying heat to prevent condensation, and by diluting vaporized solvent with a dry gas.
Liquid chromatography effluents introduced into an interface may also be incompatible with efficient electrospray ionization, particularly at high flow rates. For example, sample solutions often contain high concentrations of trifluoroacetic acid, which makes it difficult to maintain stable electrospray.
Problems also exist when coupling lower flow rates, such as those associated with capillary electrophoresis with a mass spectrometry system. Capillary electrophoresis is used for a wide variety of analyses including high resolution separations of amino acids, peptides and proteins. Capillary electrophoresis employs a capillary with an electric field gradient to separate the analyte constituents, particularly ions, by differences in electrophoretic mobilities. Capillary electrophoresis detection to date has been limited by the necessity of maintaining the quality of the separation, may require use of liquid solutions which are poorly compatible with electrospray requirements. Thus, most detectors to date have been optical detectors based on UV absorbance and fluorescence emission. Structural information necessary for the correct identification of unknown analytes and their constituents cannot be obtained using traditional detectors, and off-line analysis is impractical because of the small sample volume.
Mass spectrometry is particularly suited for detection of capillary electrophoresis eluents with high sensitivity and selectivity. Although systems have been previously described for coupling capillary electrophoresis with detection by a mass spectrometer, these systems suffer from problems such as poor sensitivity, and band broadening of the separated species due to the discrepancy of flow rates between the electrophoretic separator and the flow rates required to electrospray a sample into a mass spectrometer. (See Smith et al., Anal. Chem. 60:436-441 (1988)).
The ability of a mass spectrometer system to sample ions produced by electrospray is limited by the ability of the system to accommodate non-volatile neutrals and solvent vapor. The use of larger or more efficient vacuums or extensive heating of the interface may increase the ability of the system to accommodate such contaminants. However, non-volatiles and solvent vapor ultimately reduce efficiency, especially at high flow rates.
There are various types of electrophoresis/mass spectrometry interfaces which have been tried in the art. For example, a capillary electrophoresis system may be directly interfaced with a mass spectrometry system wherein substantially all of the sample from the CE is electrosprayed. Although these systems have good sensitivity, there are inherent problems such as difficulties in maintaining electrical continuity, thus resulting in poor stability, modification of the sample by electrochemical reaction by-products, and differences in the optimum chemical conditions for capillary electrophoresis and electrospray. Moreover, typical CE buffers introduce high chemical noise and suppress ionization efficiency.
Thus, although it is desirable to combine the high separation efficiencies of capillary electrophoresis with the inherent sensitivity of mass spectrometry, low flow rates are hard to interface without excessive band spreading, and it is difficult to generate chromatographic gradients at low flow rates
Similarly, it is desirable to combine the advantages of a high flow rate chromatography system with the sensitivity of mass spectrometry. However, high flow rates are incompatible with electrospray, and known interfacing techniques are time consuming, difficult to implement, and limited in the range of flow rates which can be accommodated. Furthermore, it is difficult to preserve the liquid chromatography separation, avoid clogging problems, and avoid wasting sample.
Accordingly, a need remains for an interface apparatus and a sample analysis method that allows an electrospray ionization technique to be used with a wide range of flow rates, such as those associated with chromatography and electrophoresis separation devices, without reducing the sensitivity of the analysis, without broadening chromatographic peaks (i.e., with no dead volume), and without wasting limited sample, and without the need for additional specialized equipment.